Method for producing therapeutic exosomes from nanoelectroporation and other non-endocytic cell transfection

ABSTRACT

Therapeutic extracellular vesicles (EVs) containing high copies of functional nucleic acids and other biomolecules are produced in large quantities by laying donor cells on a surface of a chip, adding various plasmids, other transfection vectors and their combinations to a buffer on the chip, applying a pulsulatic electric field across the cells laid on top of the chip surface and plasmids/vectors buffer solution below the chip surface, and collecting the EVs secreted by the transfected cells. The chip surface has a three-dimensional (3D) nanochannel electroporation (NEP) biochip formed on it, capable of handling large quantities of the donor cells. The buffer is adapted for receiving plasmids and other transfection vectors.

TECHNICAL FIELD

The present invention relates to methods for producing therapeuticextracellular vesicles (EVs), exosomes in particular, that containfunctional messenger RNAs (mRNAs), microRNAs (miRs), short hairpin RNAs(shRNAs), proteins, and other biomolecules by non-endocytic delivery ofDNA plasmids and other vectors into donor cells in a way that the strongstimulation caused by delivery triggers donor cells to generate a largenumber of vesicles within the cell while the non-endocytic delivery ofDNA plasmids/vectors leads to fast transcription of RNAs and translationof proteins within cytoplasm, allowing those functional biomolecules tobe encapsulated in the vesicles endogenously before they are secretedout from donor cells as EVs.

BACKGROUND

Extracellular vesicles (EVs), including exosomes, microvesicles andother vesicles, are secreted by numerous cell types. In the human body,there are >10E12 EVs in 1 mL blood and they also exist in various bodyfluids. Exosomes are nano-vesicles (40-150 nm), while microvesicles havesizes varied from <100 nm to >1 micron. They contain both coding andnon-coding RNAs and their fragments, DNA fragments, proteins, and othercell related biomolecules. EVs and their biomolecule contents have beenproposed as biomarkers for disease diagnosis. In addition, they playmajor roles in cell-cell communications in tumor microenvironment andcirculation.

EVs loaded with functional RNAs and proteins have also been suggested asdrugs and drug carriers for therapeutic applications. To deliverspecific nucleic acids and/or proteins to target tissues or cell typesin vitro and in vivo requires methods that can produce EVs with eitherendogenous or exogenous therapeutic cargos.

Post-insertion of exogenous small interference RNA (siRNA) and shRNAplasmids into pre-existed exosomes by conventional bulk electroporation(BEP) has been developed in recent years. Although their therapeuticfunctions have been successfully demonstrated in several mouse modelsfor cancer and non-cancer diseases, this approach faces manylimitations. First, post-insertion of large biomolecules such as DNAplasmids, mRNAs, and proteins into nano-sized exosomes is inefficient.Secondly, the strong electric field generated by BEP would break up manyexosomes leading to a low yield of therapeutic exosomes. Furthermore,many large biomolecules such as mRNAs and proteins are difficult andexpensive to be synthesized exogenously.

It would be highly desirable if new methods can be developed, that maytransfect donor cells with DNA plasmids or other vectors to produce alarge number of exosomes or other EVs containing therapeutic RNA andprotein targets endogenously.

In a prior U.S. patent application Ser. No. 14/282,630, we developed ananochannel electroporation (NEP) biochip that can deliver DNA plasmidsor other charged particulates and molecules into individual cellsnon-endocytically with good dosage control. Herein, we demonstrate thatNEP can produce a large number of therapeutic exosomes containing highcopies of functional mRNA and microRNA targets, not achievable by theaforementioned post-insertion methods. In addition to NEP, othernon-endocytic delivery methods such as gene gun, micro/nano-injection,etc. may also achieve a similar performance if they can provide propercell stimulation and fast plasmid/vector delivery.

SUMMARY

The present invention is related to the development of new concept andmethods that DNA plasmids and other vectors can be non-endocyticallydelivered into donor cells with strong cellular stimulation such that alarge number of vesicles and transcribed RNAs as well as translatedproteins are formed within the transfected cells. Cells would secretmany extracellular vesicles (EVs) containing specific RNA and proteintargets with therapeutic functions.

To demonstrate the aforementioned design concept, a three-dimensional(3D) NEP biochip is fabricated, that can transfect many donor cells withpre-specified DNA plasmids to secret 10˜100 folds more EVs, includingexosomes, containing high copies of intact mRNA and miR targets up tomany thousands folds more than those in EVs secreted from thenon-transfected donor cells.

Some aspects of the invention are achieved by a method of producing alarge number of therapeutic extracellular vesicles (EVs) containing highcopies of functional nucleic acids and other biomolecules. Such a methodcomprises the steps of:

laying donor cells on a surface of a chip, the surface having a threedimensional (3D) nanochannel electroporation (NEP) biochip formedthereon;

adding various plasmids, other transfection vectors and theircombinations to a buffer on the chip;

applying a pulsulatic electric field across the cells laid on top of thechip surface and plasmids/vectors buffer solution below the chipsurface, resulting in strongly stimulating the cells and deliveringplasmids/vectors into cells non-endocytically; and

collecting EVs secreted by the transfected cells.

In some of these methods, the diameter of nanochannels is between 50-900nm.

In some of these methods, wherein the plasmids and vectors transcribemRNA, microRNA, shRNA, and other RNAs, and lead to translation ofproteins and other biomolecules in the transfected cells.

In some embodiments of the method, the EVs secreted by the transfectedcells contain the transcribed mRNA, microRNA, shRNA, and other RNAs, andthe translated proteins and other biomolecules.

In some of the embodiments of the method, wherein means to increase theexpression of heat shock proteins and other proteins that can promotevesicle formation and exocytosis in the transfected cells are added tothe system, wherein the means includes a thermal shock treatment of thecells, or addition of heat shock proteins in cell culture.

In some of the embodiments, means to increase the expression of proteinsthat promote exosome formation in the transfected cells are added to thesystem, wherein the means includes co-transfection of CD63, CD9 andother DNA plasmid.

In some embodiments, multiple DNA plasmids and other vectors aredelivered to the transfected cells sequentially to promoteco-localization of RNA/protein targets and EV secretion.

In some embodiments, exogenous biomolecules such as DNA plasmids, othertransfection vectors, RNAs, proteins/peptides, small molecule drugs areencapsulated within vesicles in cells and secreted out as therapeuticEVs by sequential transfection of donor cells by NEP. In some of thesecases, in addition to NEP, other cell transfection methods that providestrong stimulation to donor cells to facilite EV secretion andnon-endocytic plasmid/vector delivery for fast RNA transcription andprotein translation are used to produce therapeutic EVs with similarefficacy. In further of these cases, the other cell transfection methodsinclude, gene gun, and micro- or nanoinjection.

In some of the embodiments, the plasmids and/or other vectors aretethered on nano- or micron-sized gold or other solid particles, andthose particles are injected into donor cells under a pneumatic forceusing a gene gun to cause strong cell stimulation and non-endocyticplasmids/vector delivery.

In some of the embodiments, the plasmids and/or other vectors aretethered on a nano- or micron-sized tip array, and donor cells arepultruded by those tips to cause strong cell stimulation andnon-endocytic plasmids/vector delivery into donor cells.

Other aspects of the invention are achieved by a device for producing alarge number of therapeutic extracellular vesicles (EVs) containing highcopies of functional nucleic acids and other biomolecules, comprising: achip having a three-dimensional (3D) nanochannel electroporation (NEP)biochip and a buffer for receiving formed thereon, the buffer adaptedfor receiving plasmids and other transfection vectors.

Other aspects of the invention comprises cells transfected by any of theforegoing methods.

BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of the disclosure can be better understood with referenceto the following drawings. The components in the drawings are notnecessarily to scale, emphasis instead being placed upon clearlyillustrating the principles of the present disclosure. In the drawings:

FIG. 1 is a schematic of a 3D Nanochannel Electroporation (NEP) biochipfor donor cell transfection;

FIG. 2A shows a comparison of cell images of BEP and NEP based celltransfection at 1 hr post-transfection using Yoyo-1 fluorescencelabelled Achaete-Scute Complex Like-1 (Ascl1) DNA plasmid, a neuronalrelated gene;

FIG. 2B shows a graphical comparison of the fluorescence intensity inthe images of FIG. 2A;

FIG. 3 shows that NEP cell transfection with or without DNA plasmidssignificantly stimulates the EV secretion from transfected mouseembryonic fibroblast (MEF) cells, with performance much better thanlipofectamine (Lipo) and BEP based cell transfection. Ctrl stands fornon-transfected MEF cells; NEP stands for NEP cell transfection with DNAplasmids; NEP-PBS stands for NEP cell transfection with PBS buffer only.The DNA plasmids used are Achaete-Scute Complex Like-1 (Ascl1), PouDomain Class 3 Transcription factor 2 (Pou3f2 or Brn2 ) and MyelinTranscription Factor 1 Like (Mytl1) at a weight ratio of 2/1/1. Amixture of those DNA plasmids is known to reprogram donor cells intoinduced neurons (iNs). The same number of MEF cells were transfectedwith DNA plasmids by various techniques, and cell culture mediums werecollected 24 h post-transfection. The EV numbers were detected byNanoSight™. For the BEP, the transfection voltage was 1250 volts; forNEP, the transfection voltage was 220 volts with five 10 ms pulses;

FIG. 4 shows the effect of heat shock protein 70 (HSP70) and heat shockprotein 90 (HSP90 ) inhibitors on EV secretion from NEP transfected MEFcells. After NEP transfection, the cell culture was replaced with freshmedium containing HSP70 inhibitor (VER 155008, 50μM), HSP90 inhibitor(NVP-HSP990, 1 μM), or their mixture. Medium was collected at 24 hpost-transfection, and EV numbers were detected by dynamic lightscattering (DLS) goniometry;

FIG. 5 shows the effect of NEP transfection of CD63 DNA plasmid on EVsecretion from MEF cells. Cells were transfected with or without CD63plasmid by NEP. The cell culture medium was collected and replaced withfresh medium every 4 hr. The EV numbers were detected by DLS goniometry;

FIG. 6A shows a size distribution measured by DLS goniometery of EVswithout NEP harvested at 24 h post-cell transfection;

FIG. 6B shows a size distribution measured by DLS goniometery of EVswith NEP harvested at 24 h post-cell transfection, where the cellculture medium was collected 24 h post transfection, where cell debriswas removed by centrifugation at 1500 g for 10 min, and the EVs insupernatant were measured by DLS;

FIGS. 7A and 7B demonstrate EV Ascl1 mRNA expression determined byqRT-PCR from MEF cells transfected by Ascl1/Brn2/Myt1l DNA plasmids at aratio of 2/1/1 using various techniques at 24 h post-transfection, withtotal RNAs obtained and reverse transcript, according to manufacturer'sinstructions, where the same amount of total RNA (20 ng) used for Ascl1detection by qRT-PCR;

FIG. 8 shows FIGS. 8A and 8B show EV Brn2 mRNA expression determined byqRT-PCR from MEF cells transfected by Ascl1/Brn2/Myt1l DNA plasmids at aratio of 2/1/1 using various techniques at 24 h post-transfection, withtotal RNAs obtained and reverse transcript, according to manufacturer'sinstructions, where the same amount of total RNA (20) used for Ascl1detection by qRT-PCR;

FIGS. 9A and 9B show EV Myt1l mRNA expression determined by qRT-PCR fromMEF cells transfected by Ascl1/Brn2/Myt1l DNA plasmids at a ratio of2/1/1 using various techniques at 24 h post-transfection, with totalRNAs obtained and reverse transcript, according to manufacturer'sinstructions, where the same amount of total RNA (20) used for Ascl1detection by qRT-PCR;

FIG. 10 shows that only EVs obtained by NEP contain functional mRNAdetermined by in vitro translation, where the same amount of total RNAs(1 μg) from each transfection group was used for in vitro proteintranslation according to manufacturer's instruction, and where thesamples were separated by SDS-PAGE, and the proteins were detected withantibodies;

FIG. 11 shows that EV-mRNAs from NEP are found in exosomes (Exo), andnot in microvesicles (MV). EVs were collected from cell culture mediumby simple centrifugation at 1500 g for 10 min. Microvesicles wereharvested by ultracentrifugation at 10,000 g for 30 min. The supernatantwas further centrifugated at 100,000 g for 2 h to collect the exosomes.Total RNAs were collected from these two parts and the total mRNAconcentration was measured by Nanodrop™. The EV mRNA expressions weremeasured by qRT-PCR.

FIG. 12 shows that exosome-mRNAs, not microvesicle-RNAs, from NEP celltransfection can translate proteins. EVs were collected from cellculture medium by simple centrifugation at 1500 g for 10 min.Microvesicles were harvested by ultracentrifugationat 10,000 g for 30min. The supernatant was further centrifugated at 100,000 g for 2 h tocollect the exosomes. Total RNAs were collected from these two parts and1 pg of total RNA was used for in vitro translation. The samples wereseparated by SDS-PAGE, and the proteins, exosomes and micrvesiclemarkers were detected by Western blotting.

FIG. 13 shows EV-mRNAs secretion profiles from NEP transfected MEFcells, where MEF cels were transfected with DNA plasmids by NEP, wherethe cell culture medium was collected at indicated time points, andreplaced with fresh medium. The EV numbers were detected by DLDgoniometry and the mRNA expressions were detected by qRT-PCR;

FIG. 14 depicts action potential detection by patch clamp shows that MEFcells transfected every other day with Ascl1/Brn2/Myt1l mRNA containingEVs obtained from NEP could be reprogrammed into functional inducedneurons (iNs) after 24 days. NEP-transfected MEF cells were reprogrammedinto iNs after 21 days.

FIGS. 15A and 15B show EV miR-128 expression determined by qRT-PCR fromMEF cells transfected by miR-128 DNA plasmid using various techniques at24 h post-transfection, where EVs were harvested from cell culturemedium at 24 h post tranfection (miR-128 plasmid) by various techniques,total RNAs were obtained according to manufacturer's instructions, andthe same amount of total RNA (30 ng) was used for miR-128 detection byqRT-PCR;

FIGS. 16A to 16E compare secreted EVs containing miR-128 by NEPtransfection of DNA plasmid to MEF cells vs. existing EVs loaded withpre-collected miR-128 by BEP post-insertion;

FIGS. 17A to 17C compare secreted EVs containing Brn2 mRNA by NEPtransfection of DNA plasmid to MEF cells vs. existing EVs loaded withpre-collected Brn2 mRNA by BEP post-insertion; and

FIG. 18 shows increased mRNA co-localization in the same EV bysequential-NEP. For NEP transfection, Ascl1, Brn2 and Myt1I plasmidswere transfected at the same time as described before. Forsequential-NEP, the Myt1l plasmid was transfected first, Brn2 plasmidwas transfected 4 h later, while Ascl1 plasmid was transfected 4 h afterBrn2 transfection. At 24 h post Myt1l transfection, culture medium wascollected for TLN assay. Equal amount of FAM-Ascl1, Cy3-Brn2, andCy5-Myt1l MBs were encapsulated in tethered lipoplex nanoparticles forEV-mRNA detection. Yellow arrow: EVs containing 3 mRNAs; Blue arrow: EVscontaining 2 mRNAs; and Pink arrow: EVs containing 1 mRNA.

DETAILED DESCRIPTION Example 1-3D NEP Biochip Schematic and Comparisonof EV Secretion and EV mRNA Content Using Different Transfection Methods

FIG. 1 shows the schematic of a 3D NEP biochip with a single layer ofdonor cells laid on the chip surface. After overnight cell incubation,the DNA plasmids pre-loaded in PBS buffer were injected into individualdonor cells via nanochannels using a 220 volts electric field across thenanochannels. Various electroporation conditions such as voltage level,pulse number and pulse length can be chosen.

Using Yoyo-1 fluorescence dye labelled Achaete-Scute Complex Like-1(Ascl1) DNA plasmid, FIGS. 2A and 2B show transfected cells imaged usingfluorescence microscopy 1 h after transfection by either BEP or NEPunder a wavelength of 488 nm. The fluorescence intensity was calculatedby NIS software. Comparison of fluorescence intensity in these twogroups is given as bar charts. The results show that BEP at themanufacturer recommended best conditions could deliver nearly 3 foldsmore plasmids than NEP at 220 volts with five 10-ms pulses to the MEFcells. However, most plasmids were still near the cell surface 1 h afterBEP transfection, while the injected plasmids by NEP have already beenuniformly diffused within cytoplasm at the same time. This implies thatBEP based cell transfection relies mainly on electroporation-mediatedendocytosis, while NEP based cell transfection is non-endocytic.

FIG. 3 compares EV numbers secreted from the same number of MEF cells(5E6 cells) transfected with the same Ascl1, Brn2 and Myt1l DNA plasmidsat a weight ratio of 2/1/1 by either lipofectamine (Lipo), BEP or NEP.All EVs were collected from cell culture medium at 24 hpost-transfection and the total EV number was determined by NanoSight™.For BEP, the transfection voltage was 1250 v with one 30-ms pulse. ForNEP, the transfection voltage was 220 with five 10 ms pulses. Theconcentration of plasmid used was Ascl1/Brn2/Myt1l=200/100/100 ng/μl.For lipofectamine transfection, 5 μg plasmid mixture(Ascl1/Brn2/Myt1l=2/1/1) was used according to manufacturer'sinstruction. The EVs were collected from cell culture medium by simplycentrifugation at 1500 g for 10 mins. The results show thatlipofectamine (Lipo) based cell transfection did not change the EVsecretion. The EV concentration was around 2E9/ml with or withouttransfection. Apparently, a slow plasmid endocytosis process bynanoparticle carriers would not stimulate the transfected cells muchand, consequently, there was almost no change on EV secretion. Incomparison, BEP based cell transfection led to more EV secretion to˜6E9/ml. A tremendous increase of EV secretion to >1.3E11/ml wasobserved by NEP cell transfection with or without adding plasmids. Thisimplies that the transfected cells were somewhat stimulated by BEP, buthighly stimulated by NEP leading to very significant increase of EVs inthe latter case.

During electroporation, Joule heating caused by the imposed electricfield could tentatively increase the cell temperature to cause thermalshocking to the transfected cells. It is known that thermal shocking mayincrease cell secretion of EVs due to chaperone mediated autophagecaused by the increase of heat shock proteins in cells (8-10). Indeed,we found that NEP could substantially increase the expression of bothheat shock protein 70 (HSP70) by 13.8 folds and heat shock protein 90(HSP90) by 4.2 folds in the transfected MEF cells vs. thenon-transfected MEF cells (Ctrl). When HSP inhibitors were added in cellculture medium after electroporation, EV secretion could be suppressed.FIG. 4 shows 50%, 40% and 70% decrease of EV secretion of NEPtransfected MEF cells with HSP 70 inhibitor (VER 155008, 50 μM) HSP90inhibitor (NVP-HSP990, 1 μM), and their mixture respectively. Here, thecell culture was replaced with fresh medium containing HSP70 inhibitor(VER 155008), HSP90 inhibitor (NVP-HSP990), or their mixture right afterNEP transfection. Medium was collected at 24 h post-transfection and EVnumbers were detected by dynamic light scattering (DLS) goniometry.These results imply that any cell stimulation that can increase theexpression of heat shock proteins would enhance EV secretion.

Similarly, an increase of proteins that are needed for late endosomalmulti-vescular body (MVB) formation in cells may also enhance exosomesecretion. FIG. 5 shows the effect of NEP transfection of CD63 DNAplasmid on EV secretion from MEF cells. Cells were transfected with orwithout CD63 DNA plasmid by NEP. The cell culture medium was collectedand replaced with fresh medium every 4 h. The EV numbers were detectedby DLS goniometry. The results show a similar EV secretion profileduring the first 16 h after NEP transfection in both cases. However,more EVs were secreted between 16 to 44 h after NEP transfection withCD63 DNA plasmid. CD63 protein is essential for the reorganization ofendosomal membrane into tetraspanin enriched microdomains, a precursorof exosome secretion.

FIGS. 6A and 6B show the EV size distribution measured by DLS goniometryfor MEFs (ctrl) and NEP transfected MEFs. NEP stimulation did not changethe larger EV (mostly microvesicles) distribution much, butsubstantially increased the secretion of exosomes with sizes rangingfrom 40 to 110 nm.

FIGS. 8A, 8B, 9A and 9B show that the secreted EVs from NEP celltransfection of Ascl1, Brn2 and Myt1l DNA plasmids contain a largeamount of corresponding Ascl1, Brn2 and Myt1l mRNAs or their fragmentsas determined using quantitative-Reverse Transcription Polymerase ChainReaction (qRT-PCR). Like the EV numbers, lipofectamine (Lipo) based celltransfection did not change the mRNA expression much, while the BEPbased cell transfection could increase the mRNA expression severalfolds. In comparison, the NEP based cell transfection resulted inthousands folds increase of target mRNAs. Here, the same amount of totalRNAs were obtained and reverse transcription was conducted by qRT-PCRaccording to manufacturer's instruction.

FIG. 10 shows that some of the EV mRNAs were intact and functionalbecause they were able to translate Ascl1, Brn2 and Myt1l proteins.Here, a same amount of total RNA (1 μg) from each transfection methodwas applied for in vitro protein translation using Rabbit ReticulocyteLysate System (Promega) according to manufacturer's instruction. Sampleswere separated by SDS-PAGE and the proteins were detected with variousantibodies as shown in the Western blotting plot.

For the collected total EVs, the larger microvesicles were sorted byultracentifugation at 10,000 g for 30 min. The supernatant was furthercentrifugated at 100,000 g for 2 h to collect the smaller exosomes.Total RNAs were collected from these two parts as described above. Thetotal mRNA concentration was measured by Nanodrop™, while the ABMexpressions of Ascl1, Brn2 and Myt1l mRNAs were measured by qRT-PCR.FIG. 11 shows that there was more than twice RNA in exosomes than inmicrovesicles, but most Ascl1, Brn2 and Myt1l mRNAs were presented onlyin exosomes. FIG. 12 shows that the functional Ascl1, Brn2 and Myt1lmRNAs were also presented in exosomes and those exosomes carry typicalexosomal protein markers, CD9, CD63 and Tsg101. In comparison, thelarger microvesicles carry the typical protein marker, Arf6.

FIG. 13 shows the EV secretion and content profiles as a function oftime after NEP transfection with Ascl1, Brn2 and Myt1l DNA plasmids. TheAscl1 plasmid is the smallest one (7 k bp) among the three, while theMyt1l plasmid is the largest (9 k bp) with the Brn2 plasmid in between(8 k bp). EVs in the cell culture medium was collected at the indicatedtime points, and the culture medium was replaced with fresh medium. TheEV numbers were detected by DLS goniometry, while the EV mRNAexpressions were detected by qRT-PCR as described before. The resultsshow a quick increase of EV secretion within 4 h post-transfection, andpeaked at 8 h with continuous EV secretion for more than 24 h. EVscontaining Ascl1 and Brn2 mRNAs also appeared within 4 hpost-transfection with profiles matching well with that of EV secretion.EVs containing Myt1l mRNA appeared at a later time, but still within 24h. This implies that the EV secretion time and the mRNA transcriptiontime need to be matched in cell, which can be achieved by NEP based celltransfection.

To demonstrate that NEP-produced-EVs containing endogenous mRNAs havetherapeutic functions, we treated MEF cells with those EVs every otherday at a total EV RNA concentration of 1 μg per 100,000 cells. Afterseveral days, the treated MEF cells started to reveal neuron-likemorphology and at 24 days, the treated cells showed electrophysiologicalactivity as demonstrated by their capacity to undergo induced actionpotentials as shown in FIG. 14 . In comparison, the NEP-transfected MEFcells also showed a similar electrophysiological activity on Day 21.Cells displayed the necessary voltage-gated currents to fire actionpotentials. Both transient inward currents and sustained outwardcurrents were observed in response to depolarizing voltage simulations.A typical response to a 20 pA current injection is illustrated in FIG.14 and indicates that cells fired action potentials in response todepolarizing current.

Whole-cell patch clamp recording was used to measure excitability. Cellswere continuously superfused with an extracellular bath solutioncontaining 115 mM NaCl, 2 mM KCl, 1.5 mM MgCl₂, 3 mM CaCl₂), 10 mMHEPES, and 10 mM Glucose (pH 7.4). Glass electrodes (3-4 MΩ) were filledwith a pipette solution containing 115 mM K-gluconate, 10 mMN-2-Hydroxyethylpiperazine-N′-2-Ethanesulfonic Acid (HEPES), 4 mM NaCl,0.5 mM ethylene glycol tetraacetic acid (EGTA), 1.5 mM MgCl₂, (pH 7.3).Cells had a patch resistance of >100 MOhm after whole-cell access wasgained, and series resistance was compensated 40-50%. Data werecollected using an Axopatch 200B amplifier, Digidata 1322A digitizer,and Clampex 9 software (Molecular Devices, Sunnyvale, Calif.). Foranalysis of voltage-gated currents, the basal holding potential was −70mV and cells were stepped for 400 ms in 10 mV increments from −120 mV to80 mV. Transient inward currents, due to activity of voltage-gatedsodium channels, were isolated from measuring the peak amplitude.Sustained plateau currents, reflective of voltage-gated potassiumcurrents, were measured as the average of the last 50 ms of the voltagestep in the plateau phase of the current. Action potential induction wasmeasured using current clamp. Current was held at 0 pA and then steppedin 20 pA intervals for 1 sec.

Example 2—EV MicroRNA Content Using Different Transfection Methods

To demonstrate broader therapeutic applicability, we also transfectedMEF cells with a DNA plasmid that will transcribe microRNA targets incells. FIGS. 15A and 15B show the EV miR-128 expression for EVsharvested from cell culture medium at 24 h post-transfection (miR-128plasmid) by various techniques. Total RNAs were obtained according tomanufacturer's instruction. The same amount of total RNA (30 ng) wasused for miR-128 detection by qRT-PCR using the aforementionedprocedures. Again, NEP based transfection was able to produce EVscontaining a large amount of miR-128 (more than 4,500 folds increase),not achievable by BEP or lipofectamine based cell transfection.

Example 3—Comparison of EVs Containing Endogenous RNAs by NEPTransfection of DNA Plasmid to MEF Cells Vs. Existing EVs Loaded withPre-Collected RNAs by BEP Post-Insertion

Here, we compared the efficacy of producing therapeutic EVs using ourNEP based cell transfection and the BEP post-insertion approach used byseveral researchers. For the former, the miR-128 plasmid wasco-transfected with CD63-GFP plasmid to MEF cells by NEP to generate EVscontaining miR-128 according to aforementioned procedures. For thelatter, blank EVs were first harvested from MEF cells transfected withCD63-GFP plasmid 24 h after NEP. In parallel, miR-128 was collected fromMEF cells transfected with miR-128 plasmid 24 h post-transfection byNEP. The collected miR-128 (1 μg) was mixed with blank EVs (10E6) andelectroporated by BEP (1250 volts, 30 ms) according to conditions usedby other researchers. EVs from the two approaches were tested using atethered lipoplex nanoparticle (TLN) biochip on a total internalreflection fluorescence (TIRF) microscope. FIG. 16A shows the TLN-TIRFassay schematic (2, 11). Briefly, a molecular beacon (MB) for the RNAtarget is designed and encapsulated in cationic liposomal nanoparticles.These cationic lipoplex nanoparticles are tethered on a glass slide,which are able to capture negatively charged EVs by electrical staticinteractions to form a larger nanoscale complex. This lipoplex-EV fusionleads to mixing of RNAs and MBs within the nanoscale confinement nearthe biochip interface. TIRF microscopy is capable of detecting a singlebiomolecule and it measures signals <300 nm near the interface, which iswhere the tethered liposomal nanoparticles locate.

FIG. 16B shows the representative TLN-TIRF images of the captured EVs.The green fluorescence is from EVs containing CD63-GFP, while the redfluorescence is from hybridization of miR-128 molecules and theCy5-miR128 MBs in the captured EVs. It is clear that our NEP approach isable to produce more EVs containing higher copies of miR-128 than theBEP post-insertion approach. FIGS. 16C-E show a quantitative comparisonof those two approaches. Although both approaches are able to produceEVs containing miR-128 (˜80% of total captured EVs), the EV miR-128concentration in EVs (˜3 times MB fluorescence intensity) is much higherin NEP based direct cell transfection than in BEP based microRNApost-insertion. Furthermore, BEP post-insertion tends to break nearlyhalf of the blank EVs leading to a very low yield of therapeutic EVs.

A similar comparison was also carried out for a much larger RNA, Brn2mRNA (6272 bases for Brn2 mRNA vs. 21 bases for miR-128) using the sameapproach as for miR-128. FIGS. 17A to 17C show that our NEP approachcould produce >70% EVs containing Brn2 mRNA, while only very fewexisting EVs could be loaded with the same mRNA by BEP post-insertionapproach. The concentration of Brn2 mRNA in NEP produced EVs is high,while that in BEP post-insertion is very poor.

Example 4—Improvement of Multiple mRNAs Co-Localized in the SameSecreted EVs by Sequential NEP Transfection of DNA Plasmids to MEF Cells

FIG. 13 implies that different mRNA targets could be transcribed atdifferent times and rates in the transfected cells, even though multipleDNA plasmids were delivered to the cells at the same time, due to thesize difference of plasmids or other reasons. This may lead toindividual EVs containing only one or few mRNA targets. For bettertherapeutic efficacy, it would be valuable if more or all mRNA targetscan be encapsulated in the same secreted EVs. By sequentially deliveringeach DNA plasmid into MEF cells using NEP based on its transcriptiontime, FIG. 18 shows that we could substantially increase the secretedEVs containing all three mRNAs, Ascl1, Brn2 and Myt1l (>50% vs. <25%),needed for iN reprogramming. For NEP transfection, Ascl1, Brn2 and Myt1lplasmids were transfected at the same time as described before. Forsequential-NEP, the Myt1l plasmid was transfected first, Brn2 plasmidwas transfected 4 h later, while Ascl1 plasmid was transfected 4 h afterBrn2 transfection. At 24 h post Myt1l transfection, culture medium wascollected for TLN assay. Equal amount of FAM-Ascl1, Cy3-Brn2, andCy5-Myt1l MBs were encapsulated in tethered lipoplex nanoparticles forEV-mRNA detection. In the figure, the yellow arrow means EVs containingall 3 mRNAs, the blue arrow means EVs containing 2 mRNAs, while the pinkarrow means EVs containing only 1 mRNA.

While the invention has been explained in relation to its preferredembodiments, it is to be understood that various modifications thereofwill become apparent to those skilled in the art upon reading thespecification. Therefore, it is to be understood that the inventiondisclosed herein is intended to cover such modifications as fall withinthe scope of the appended claims.

What is claimed is:
 1. A method of producing extracellular vesicles(EVs) comprising at least one RNA transcribed from DNA, comprising: (a)providing an electroporation biochip, the electroporation biochipcomprising: (i) donor cells in contact with a first surface of theelectroporation biochip and secreting a baseline amount of secretedextracellular vesicles (EVs);(ii) a buffer comprising the DNA, whereinthe buffer comprising the DNA is in contact with a second surface of theelectroporation biochip; and (iii) a plurality of channels that connectthe first surface of the electroporation biochip with the second surfaceof the electroporation biochip, wherein the plurality of channels arepositioned such that individual channels of the plurality of channelscontacts individual donor cells of the donor cells in contact with thefirst surface of the electroporation biochip; (b) applying a pulsedelectric field to the electroporation biochip wherein the pulsedelectric field is applied as a plurality of pulses wherein each pulse isat least one millisecond in length and such that the pulsed electricfield delivers the DNA to the donor cells to produce transfected donorcells, wherein the pulsed electric field causes the transfected donorcells to secrete 10-fold to 100-fold more extracellular vesicles (EVs)compared to the baseline amount of secreted extracellular vesicles (EVs)and wherein the transfected donor cells release extracellular vesicles(EVs) comprising the at least one RNA transcribed from the DNA; and (c)collecting the extracellular vesicles (EVs) comprising the at least oneRNA transcribed from the DNA.
 2. The method of claim 1, wherein thebuffer comprising the at DNA comprises a DNA plasmid, DNA vector, orcombination thereof and the transfected donor cells comprise the DNAplasmid, DNA vector, or combination thereof.
 3. The method of claim 2,wherein the extracellular vesicles (EVs) comprise at least one RNAtranscribed from the DNA plasmid, DNA vector, or combination thereof. 4.The method of claim 1, wherein the at least one RNA transcribed from theDNA, is at least one RNA selected from the group consisting of:messenger RNA (mRNA), non-coding RNA, microRNA (miRNA), short hairpinRNA (shRNA), and any combination thereof.
 5. The method of claim 1,wherein the at least one RNA transcribed from the DNA comprisesmessenger RNA (mRNA).
 6. The method of claim 1, wherein a DNA plasmid orDNA vector encoding CD63, CD9, or combination thereof, is delivered intothe transfected donor cells.
 7. The method of claim 1, furthercomprising delivery of a biomolecule or therapeutic drug to thetransfected donor cells by an additional method of transfection.
 8. Themethod of claim 7, wherein the additional method of transfection isselected from the group consisting of gene gun, microinjection, andnanoinjection.
 9. The method of claim 8, wherein the additional methodof transfection is microinjection or nanoinjection, and the biomoleculeor therapeutic drug is tethered on a nano- or micron-sized tip array.10. The method of claim 1, wherein the donor cells are present in asingle layer on the first surface of the electroporation biochip. 11.The method of claim 1, wherein a portion of the extracellular vesicles(EVs) comprise messenger RNA (mRNA) transcribed from the DNA.
 12. Themethod of claim 11, wherein at least 70% of the extracellular vesicles(EVs) comprise messenger RNA (mRNA) transcribed from the DNA.
 13. Themethod of claim 11, wherein at least 25% of the extracellular vesicles(EVs) comprise messenger RNA (mRNA) transcribed from the DNA.
 14. Themethod of claim 1, wherein the extracellular vesicles (EVs) compriseexosomes.
 15. The method of claim 14, wherein the exosomes comprise CD9protein marker, CD63 protein marker, Tsg101 protein marker, or acombination thereof.
 16. The method of claim 1, further comprisingculturing the donor cells on the first surface of the electroporationbiochip for a length of time prior to (b).
 17. The method of claim 16,wherein the culturing length of time is overnight.
 18. The method ofclaim 1, wherein the at least one RNA transcribed from the DNA comprisesat least one mRNA of at least 21 bases and up to 7144 bases.
 19. Themethod of claim 1, wherein the at least one RNA transcribed from the DNAcomprises between 21 and 6272 bases.
 20. The method of claim 1, whereinthe extracellular vesicles (EVs) are collected at least 4 hours up to 24hours after (b).
 21. The method of claim 1, wherein the at least one RNAtranscribed from the DNA is intact and is capable of being translatedinto one or more peptides in vitro.
 22. The method of claim 1, whereinthe transfected donor cells release extracellular vesicles (EVs) in anamount that is three-fold higher than a concentration of extracellularvesicles (EVs) released had the transfected donor cells been transfectedusing lipofectamine.
 23. The method of claim 1, wherein the donor cellsare situated in a first compartment that comprises the first surface ofthe electroporation biochip.
 24. The method of claim 23, wherein thefirst vessel compartment that comprises the first surface of theelectroporation biochip is situated in a second compartment thatcomprises the buffer comprising the DNA.
 25. The method of claim 24,wherein the first compartment is fluidly connected to the secondcompartment via the plurality of channels.
 26. The method of claim 1,wherein the extracellular vesicles (EVs) are collected up to 24 hoursafter (b).